Gene expression and in vitro nuclear maturation in bovine cumulus oocyte complexes maturated in a medium supplemented with bovine fetal serum or bovine serum albumin

ABSTRACT

Background: The expansion and mucification of granulosa cells of the cumulus oophorus-oocyte complex (COC) is observed during the oocyte in vitro maturation (IVM) as a result of the intense synthesis of extracellular matrix (ECM) components. These changes in cumulus aspect are indicative of maturation and may be infl uenced by oocyte-related factors and by IVM conditions. The objectives of the present study were (i) to assess the expression of gene transcripts that codify for the proteins hyaluronan synthase-2 (HAS2), link protein 1, connexin 43 and ß-actin in bovine cumulus oophorus-oocyte complexes (COCs) before and after IVM, and (ii) to determine nuclear maturation rates of oocytes submitted to IVM. Materials, Methods & Results: Bovine COCs obtained from abattoir-derived ovaries were analyzed and selected for morphological aspects and divided in three experimental groups G1, COCs submitted to IVM; G2, COCs submitted to IVM in medium supplemented with 10% fetal bovine serum (FBS); and G3, COCs submitted to IVM in medium supplemented with bovine serum albumin (BSA). After extraction of the messenger RNA (mRNA) of COCs, cDNA was extracted and fragments of the gene transcripts were amplified using the reverse transcription (RT) and the polymerase chain reaction (PCR). The RT-PCR products were electrophoresed in agarose gels and amplification intensity was quantifi ed to obtain the relative mRNA abundance. Part of oocytes submitted to IVM medium supplemented with FBS (G2) or BSA (G3) was stained with Hoechst 33342 to assess the nuclear maturation rate by fluorescence microscopy. The results revealed that relative abundances of HAS (P = 0.000), link protein 1 (P = 0.001), connexin 43 (P = 0.007) and ß-actin (P = 0.011) transcripts differed between COCs submitted to IVM in FBS medium (G2) and COCs not submitted to IVM (G1) or COCs maturated in BSA medium (G3). When COCs submitted to IVM in FBS or BSA media are compared, no statistically significant differences (P > 0.05) were observed in meiosis resumption (86.7% and 91.5%, respectively) or in nuclear maturation rates (56.1% and 58.5%). Discussion: HAS2 is involved in the synthesis of hyaluronic acid (HA) by cumulus cells, and plays an important role in ECM expansion and in oocyte competence development. This protein organization of the ECM, formed by the aggregation of HA and proteoglycans, depends on link protein 1; it is also produced by cumulus cells and is implicated in COC expansion. Connexin 43 belongs to a protein family that establishes gap junctions that play an important role in the cellular communication and coordinated response processes. The role of gap junctions in bovine oocytes during IVM has been associated with maturation rates and cumulus expansion; this expansion of cumulus cells is accompanied by changes in the transmembrane channels formed by connexin 43. The higher mRNA expression of the HAS2, link protein 1, connexin 43 and ß-actin genes in bovine COCs submitted to IVM in FBS medium, in comparison with COCs before IVM or COCs maturated in BSA medium may be associated with FBS constituents, which would act as transcription factors for these genes during ECM expansion. Although the results obtained allow associating the differential expression of transcripts to the presence of FBS in the IVM medium, the data reveal that meiosis resumption and nuclear maturation apparently were not influenced by the protein supplementation regimens in the IVM medium, supplemented either with FBS or BSA.